The goal of this research is to understand the mechanism of co-translational translocation of nascent secretory and membrane proteins in molecular detail. This process occurs at the plasma membrane in bacteria and at the ER membrane in eukaryotes. Translocation is mediated by the SecY or Sec61 channel bound to its cognate ribosome. In eukaryotes, complementary studies of the translocon associated protein complex (TRAP) will be continued and the role of the ribosome-Bag6 complex in the insertion pathway for tail-anchored membrane proteins will be investigated. In Aim 1, four structures will be determined of E. coli ribosome-SecY complexes at various stages of translocation of a secretory protein. We will use state-of-the-art cryo-EM images taken on a direct electron detector to provide high resolution 3D maps of the complexes. This will include a ground state structure without a nascent chain and structures of secretory complexes with nascent chains at early and late stages of translocation. In these studies, active 70S-SecY complexes are formed in cells and stalled nascent chains are crosslinked within the channel to trap the translocation intermediates. This Aim will reveal conformational changes of the SecY complex and the disposition of the nascent chain, as the lateral gate opens to allow a signal sequence helix to move into the surrounding bilayer and be processed by signal sequence peptidase. This cleavage step leaves a SecY channel with a single strand of the translocating nascent chain threaded across the pore. In Aim 2, the goal is to obtain a crystal structure of the alpha/beta lumenal domain of TRAP to test our hypothesis that the alpha subunit may act like a chaperone that transiently binds to extended nascent chains as they exit from the Sec61 channel. This may bias translocation in the forward direction. In Aim 3, the structure of a human Bag6-TRC35-Ubl4A complex will be determined, as it interacts with 80S ribosomes that contain a transmembrane (TM) domain in the exit tunnel. This is the first step of the insertion pathway for tail anchored membrane proteins, in which the TM helix is protected from aggregation by the Bag6 complex. The TM helix is subsequently delivered to the TRC40 ATPase dimer, which transfers its substrate to the Get1-Get2 complex for insertion into the ER membrane.